Determination of relative Notch1 and gamma-secretase-related gene expression in puromycin-treated microdissected rat kidneys.

Notch signaling pathways are involved in the regulation of cell differentiation and are highly conserved across species. Notch ligand binding leads to gamma-secretase-mediated proteolytic cleavage of the Notch receptor releasing the Notch intracellular domain, resulting in its subsequent translocation into the nucleus and gene expression regulation. To investigate the level of expression of Notch signaling pathway components in microanatomic regions following renal injury, kidneys from untreated, vehicle control, and puromycin aminonucleoside (PA, 150 mg/kg)-treated rats were evaluated. Frozen tissue sections from rats were microdissected using laser capture microdissection (LCM) to obtain glomeruli, cortical (proximal) tubules, and collecting ducts, and relative gene expression levels of Presenilin1, Notch1 and Hes1 were determined. In untreated rats, the Notch1 expression in glomeruli was higher than in the proximal tubules and similar to that in collecting ducts, whereas Presenilin1 and Hes1 expressions were highest in the collecting ducts, followed by cortical tubules and glomeruli. Following PA-induced renal injury, Hes1 gene expression increased significantly in the glomeruli and tubules compared to the collecting ducts where no injury was observed microscopically. Although these data present some evidence of change in Notch signaling related to injury, the expression of Presenilin1, Notch1, and Hes1 in the microanatomic regions of the kidney following PA treatment were not significantly different when compared to controls. These results demonstrate that there are differences in Notch-related gene expression in the different microanatomic regions of the kidneys in rats and suggest a minimal role for Notch in renal injury induced by PA. In addition, this work shows that LCM coupled with the RT-PCR can be used to determine the relative differences in target gene expression within regions of a complex organ.

Assessing cell fusion and cytokinesis failure as mechanisms of clone 9 hepatocyte multinucleation in vitro.

In this in vitro model of hepatocyte multinucleation, separate cultures of rat Clone 9 cells are labeled with either red or green cell tracker dyes (Red Cell Tracker CMPTX or Vybrant CFDA SE Cell Tracer), plated together in mixed-color colonies, and treated with positive or negative control agents for 4 days. The fluorescent dyes become cell-impermeant after entering cells and are not transferred to adjacent cells in a population, but are inherited by daughter cells after fusion. The mixed-color cultures are then evaluated microscopically for multinucleation and analysis of the underlying mechanism (cell fusion/cytokinesis). Multinucleated cells containing only one dye have undergone cytokinesis failure, whereas dual-labeled multinucleated cells have resulted from fusion.